Phytochemistry 65 (2004) 249–259
www.elsevier.com/locate/phytochem
Molecules of interest
Horseradish peroxidase: a modern view of a classic enzyme
Nigel C. Veitch*
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
Abstract
Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years
new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms
of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now
used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered
enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A
combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in
targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism,
cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of
specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the
identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Horseradish peroxidase; Armoracia rusticana; Arabidopsis thaliana; Cruciferae; Heme; Hydrogen peroxide; Indole-3-acetic acid; Protein
engineering
1. Introduction
The horseradish (Armoracia rusticana P.Gaertn.,
B.Mey. & Scherb.; Cruciferae) is a hardy perennial herb
cultivated in temperate regions of the world mainly for
the culinary value of its roots (Fig. 1). These are also a
rich source of peroxidase, a heme-containing enzyme
that utilises hydrogen peroxide to oxidise a wide variety
of organic and inorganic compounds. Production of
peroxidase from horseradish roots occurs on a relatively
large scale because of the commercial uses of the
enzyme, for example as a component of clinical diagnostic kits and for immunoassays. Although the term
horseradish peroxidase is used somewhat generically,
the root of the plant contains a number of distinctive
peroxidase isoenzymes of which the C isoenzyme (HRP
C) is the most abundant. This has been the subject of
much of the published work on horseradish peroxidase,
which comprises many thousands of papers in the
scientific literature. Some major advances in our understanding of the structure and function of HRP C have
* Corresponding author. Tel.: +44-20-8332-5312; fax: +44-208332-5310.
E-mail address: n.veitch@rbgkew.org.uk (N.C. Veitch).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2003.10.022
been achieved relatively recently, and were initiated by
the successful production of recombinant enzyme
(Smith et al., 1990). From this work came the longawaited solution of the three-dimensional structure of
HRP C by X-ray crystallography and more recently, a
high resolution description of the intermediates in the
catalytic cycle of the enzyme (Gajhede et al., 1997; Berglund et al., 2002). Many physiological roles have been
assigned to horseradish peroxidase isoenzymes including indole-3-acetic acid metabolism, lignification, crosslinking of cell wall polymers, suberin formation and
resistance to infection. It is surprising therefore that so
little is known about their specific functions in the plant.
New insights into this problem may result from comparative studies of the peroxidases of the ‘model plant’,
Arabidopsis thaliana (L.) Heynh., which is also a member of the Cruciferae. Two groups have now described
the entire peroxidase gene family of A. thaliana, which
comprises no less than 73 full-length genes, the majority
of which are predicted to code for stable enzymes
(Tognolli et al., 2002; Welinder et al., 2002). It seems
therefore, that each plant species contains a ‘suite’ of
peroxidase isoenzymes with the potential to carry out a
range of different functions. This is one of the issues
that will be highlighted in the present paper, which aims
to provide an overview of recent advances in our
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N.C. Veitch / Phytochemistry 65 (2004) 249–259
Fig. 1. An early illustration of the horseradish plant, from the 1633
edition of Gerard’s celebrated ‘Herball, or generall historie of plantes’.
According to Gerard ‘the root is long and thick, white of colour, in
taste sharpe, and very much biting the tongue like mustard’. These
properties are due to volatile isothiocyanates released by the hydrolysis of constituent glucosinolates when the roots are crushed, a reaction that is catalysed by myrosinase. The same roots are also one of
the most important sources of the heme-containing enzyme, peroxidase. Reproduced with permission. Copyright 2003, Royal Botanic
Gardens, Kew.
blue product also known as ‘guaiacum blue’ (Kratochvil
et al., 1971). Although the term ‘peroxidase’ came into
use towards the end of the nineteenth century it was the
work of Robert Chodat (1865–1934) and Alexei Nikolaevich Bach (1857–1946) at the University of Geneva
during the early years of the twentieth century that
initiated further research on peroxidase from the horseradish root and other plant sources (Bach and Chodat,
1903). Some of their observations stimulated a vigorous
debate on the nature of oxidases and peroxidases which
was later recounted in a book by David Keilin (1887–
1963), another distinguished contributor to the peroxidase field (Keilin, 1966). Although Bach and Chodat
obtained a crude preparation of horseradish peroxidase
it was through the subsequent studies of Richard
Willstätter (1872–1942) and Hugo Theorell (1903–1982)
that pure enzyme was finally isolated. Other advances in
the period from approximately 1920 to 1945 included
the demonstration of heme and carbohydrate as components of horseradish peroxidase, the first observation
of the catalytic intermediates known as compounds I
and II and the first kinetic analysis of the reaction with
hydrogen peroxide. Detailed commentaries on the
history and development of peroxidase are available in
the literature (Saunders et al., 1964; Paul, 1986). These
emphasise the important contribution that research on
horseradish peroxidase has made to our understanding
of peroxidase enzymes in general.
3. Description of the enzyme
3.1. General features
understanding of horseradish peroxidase and a
summary of its most important characteristics and uses.
2. Historical perspectives
The first recorded observation of a reaction catalysed
by horseradish peroxidase appears in a note published
by Louis Antoine Planche (1776–1840) describing the
analysis of ‘jalap’ resin imported to France for medicinal use, probably from the Carribean (Planche, 1810).
A series of tests was carried out to determine whether a
particular consignment was adulterated with ‘gaiac’
resin (also known as ‘guaiacum’, the powdered heartwood of the Carribean trees, Guaiacum officinale and G.
sanctum). During these investigations Planche noted
that a tincture of gaiac resin became a beautiful blue
colour when a piece of fresh horseradish root was
placed in it. The reaction observed was probably the
peroxidase-catalysed oxidation of 2,5-di-(4-hydroxy-3methoxyphenyl)-3,4-dimethylfuran (a minor constituent
of the resin and formerly referred to as a-guaiaconic
acid) to give the corresponding bis-methylenequinone, a
Horseradish peroxidase isoenzyme C comprises a single polypeptide of 308 amino acid residues, the sequence
of which was determined by Welinder (1976). The Nterminal residue is blocked by pyroglutamate and the Cterminus is heterogenous, with some molecules lacking
the terminal residue, Ser308. There are 4 disulphide
bridges between cysteine residues 11–91, 44–49, 97–301
and 177–209, and a buried salt bridge between Asp99
and Arg123. Nine potential N-glycosylation sites can be
recognised in the primary sequence from the motif AsnX-Ser/Thr (where ‘X’ represents an amino acid residue)
and of these, eight are occupied. A branched heptasaccharide accounts for 75 to 80% of the glycans
(Table 1) but the carbohydrate profile of HRP C is
heterogeneous, and many minor glycans have also been
characterised (Yang et al., 1996). These invariably contain two terminal GlcNAc and several mannose residues. A further complication is the variation in the type
of glycan present at any of the glycosylation sites. The
total carbohydrate content of HRP C is somewhat
dependent on the source of the enzyme and values of
between 18 and 22% are typical.
N.C. Veitch / Phytochemistry 65 (2004) 249–259
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Table 1
Anatomy of a peroxidase. Some essential structural features of horseradish peroxidase isoenzyme C (HRP C)
Heme
(i)
(ii)
His170 forms coordinate bond to heme iron atom.
Asp247 carboxylate side-chain helps to control
imidazolate character of His170 ring.
(iii) His170Ala mutant undergoes heme degradation
when H2O2 added and compounds I and II are not
detected; k1 = 1.6101 M1 s1. Imidazoles can
bind to heme iron in the artificially created cavity
but full catalytic activity is not restored because the
His170Ala-imidazole complex does not maintain a
five-coordinate state (His42 also binds to Fe).
(iv) Aromatic substrates are oxidized at the exposed
heme edge but do not bind to heme iron.
Calcium
Distal O-donors
Proximal O-donors
Asp43, Asp50, Ser52
(side-chain)
Asp43, Val46, Gly48
(carbonyl)
1 structural water
Thr171, Asp222,
Thr225, Asp230
(side-chain)
Thr171, Thr225,
Ile228 (carbonyl)
Distal and proximal Ca2+ ions are both
seven-coordinate.
(ii) On calcium loss enzyme activity decreases by 40%.
(iii) Structural water of distal Ca site hydrogen bonded
to Glu64 which is itself hydrogen bonded to Asn70
and thus connects to the distal heme pocket.
(i)
Carbohydrate
(i)
(ii)
Sites of glycosylation are in loop regions of the
structure, at Asn13, Asn57, Asn158, Asn186,
Asn198, Asn214, Asn255 and Asn268.
The major glycan is shown here; there are also
minor glycans of the form ManmGlcNAc2
(m = 4 to 7) and (Xyl)xManm(Fuc)f GlcNAc2
(m = 2, 4, 5, 6; f = 0 or 1; x = 0 or 1).
Amino acid residues
Arg38
Essential roles in (i), the formation and stabilization of compound I,
(ii) binding and stabilization of ligands and aromatic substrates
(e.g. benzhydroxamic acid, ferulate etc.).
Arg38Leu mutant; k1 = 1.1104 M1 s1;
Kd (BHA) 12.1 0.7 mM.
Phe41
Prevents substrate access to the ferryl oxygen of compound I.
Phe41Ala/Leu/Thr mutants show improved rates
of thioanisole oxidation (oxygen-transfer chemistry).
His42
Essential roles in (i), compound I formation (accepts proton
from H2O2), (ii) binding and stabilization of ligands and
aromatic substrates.
His42Leu mutant; k1 = 1.4102 M1 s1;
Kd (BHA) 2.90.5 mM.
Asn70
Maintains basicity of His42 side-chain through Asn70-His42 couple
(hydrogen bond from Asn70 amide oxygen to His42 imidazole NH).
Asn70Val mutant; k1 = 6.0105 M1 s1.
Pro139
Part of a structural motif, ‘-Pro-X-Pro-’ (Pro139-Ala140-Pro141
in HRP C), which is conserved in plant peroxidases.
Crystallographic evidence indicates role for Pro139
in substrate oxidation and binding. Note that
residues Ala140, Phe142 and Phe143 are also part
of the ferulate binding site shown in Fig. 5.
Original references for the results described in this table can be found in Veitch and Smith (2001). The rate constant for compound I formation (k1)
for wild-type enzyme is 1.7107 M1 s1. The apparent dissociation constant (Kd) for complex formation between resting state HRP C and benzhydroxamic acid is 2.1 0.2 mM (pH 7.0, 25 C).
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HRP C contains two different types of metal centre,
iron(III) protoporphyrin IX (usually referred to as
the ‘heme group’) and two calcium atoms (Fig. 2
and Table 1). Both are essential for the structural and
functional integrity of the enzyme. The heme group is
attached to the enzyme at His170 (the proximal histidine residue) by a coordinate bond between the histidine
side-chain NE2 atom and the heme iron atom. The second axial coordination site (on the so-called distal side
of the heme plane) is unoccupied in the resting state of
the enzyme but available to hydrogen peroxide during
enzyme turnover (Fig. 3). Small molecules such as
carbon monoxide, cyanide, fluoride and azide bind to
the heme iron atom at this distal site giving six-coordinate peroxidase complexes. Some bind only in their
protonated forms, which are stabilized through hydrogen bonded interactions with the distal heme pocket
amino acid side-chains of Arg38 (the distal arginine)
and His42 (the distal histidine) (Fig. 3).
The two calcium binding sites are located at positions
distal and proximal to the heme plane and are linked to
the heme-binding region by a network of hydrogen
bonds. Each calcium site is seven-coordinate with oxygen-donor ligands provided by a combination of amino
acid side-chain carboxylates (Asp), hydroxyl groups
(Ser, Thr), backbone carbonyls and a structural water
molecule (distal site only) (Table 1). Loss of calcium
results in decreases to both enzyme activity and thermal
stability (Haschke and Friedhoff, 1978) and to subtle
changes in the heme environment that can be detected
spectroscopically (Howes et al., 2001).
Fig. 2. Three-dimensional representation of the X-ray crystal structure of horseradish peroxidase isoenzyme C (Brookhaven accession
code 1H5A). The heme group (coloured in red) is located between the
distal and proximal domains which each contain one calcium atom
(shown as blue spheres). a-Helical and b-sheet regions of the enzyme
are shown in purple and yellow, respectively. The F 0 and F 00 a-helices
appear in the bottom right-hand quadrant of the molecule.
Fig. 3. Key amino acid residues in the heme-binding region of HRP
C. The heme group and heme iron atom are shown in red, the
remaining residues in atom colours. His170, the proximal histidine
residue, is coordinated to the heme iron atom whereas the corresponding distal coordination site above the plane of the heme is
vacant. Specific functions for the amino acid residues shown here are
given in Table 1.
3.2. Three-dimensional structure
The first solution of the three-dimensional structure
of HRP C using X-ray crystallography appeared in the
literature relatively recently (Gajhede et al., 1997). The
recombinant enzyme used as the source of crystals and
heavy atom derivatives was produced by expression in
Escherichia coli in non-glycosylated form (Smith et al.,
1990). Previous attempts to obtain suitable crystals for
diffraction were frustrated partly by the heterogeneity of
plant HRP C preparations comprising multiple glycoforms. The structure of the enzyme is largely a-helical
although there is also a small region of b-sheet (Fig. 2).
There are two domains, the distal and proximal,
between which the heme group is located. These
domains probably originated as a result of gene duplication, a proposal supported by their common calcium
binding sites and other structural elements (Welinder
and Gajhede, 1993).
N.C. Veitch / Phytochemistry 65 (2004) 249–259
3.3. HRP and the plant peroxidase superfamily
Horseradish peroxidase isoenzymes belong to class
III (‘classical’ secretory plant peroxidases) of the plant
peroxidase superfamily, which includes peroxidases of
bacterial, fungal and plant origin (Welinder, 1992a).
The remaining two classes comprise yeast cytochrome c
peroxidase, gene-duplicated bacterial peroxidases and
ascorbate peroxidases (class I), and fungal peroxidases
(class II). This classification was based originally on
comparisons of amino acid sequence but is well-supported by more recent three-dimensional structural data
obtained for enzymes from each of the classes. Many
structural elements are conserved among peroxidases of
the three classes leading to the definition of a ‘core’
peroxidase fold. The structures of HRP C and of other
class III plant peroxidases contain three a-helices that
are additional to this core peroxidase fold (Gajhede et
al., 1997). Two of these (helices F 0 and F 00 ) are located
in a long insertion that shows great variation in both
sequence and number of residues (Fig. 2). The overall
integrity of the structure in this region is maintained by
the disulphide linkage from Cys177 to Cys209. What is
particularly interesting in the case of HRP C is the
location of residues in helix F 0 that are involved in substrate access and binding. Some authors have speculated
that this structural region is important for stabilization
or retention of radical species produced in reactions
catalysed by plant peroxidases (Gajhede et al., 1997). A
comparison of structural and functional characteristics
of peroxidases from the three classes of the plant
peroxidase superfamily has been published (Smith and
Veitch, 1998).
253
ascorbate peroxidase (Apx1) was demonstrated (Pnueli
et al., 2003). An interesting observation in the knockout-Apx1 plants was the induction of transcripts
encoding for two class III plant peroxidases, presumably to compensate for the reduction in peroxidescavenging ability.
The formation of radical products in HRP-catalysed
reactions gives an indication of some possible in vivo
functions in the plant. These may involve cross-linking
reactions such as the formation of diferulate linkages
from polymer-attached ferulate groups of polysaccharides or pectins, the formation of dityrosine
linkages, the cross-linking of phenolic monomers in the
formation of suberin and the oxidative coupling of
phenolic compounds as part of the biosynthesis of lignin. Peroxidases that catalyse cross-linking reactions
may be expressed in response to external factors such as
the wounding of plant tissue. Water loss and invasion
by pathogens can thus be limited by the formation of a
protective polymeric barrier such as suberin. Although
in vivo roles for HRP isoenzymes have not yet been
determined, it is known that peroxidase activity is
induced when horseradish leaves are wounded
(Kawaoka et al., 1994).
4.2. Catalytic mechanism
The mechanism of catalysis of horseradish peroxidase
and in particular, the C isoenzyme, has been investigated extensively (reviewed in Dunford, 1991, 1999;
Veitch and Smith, 2001). Some important features of
the catalytic cycle are illustrated in Fig. 4 with ferulic acid
as reducing substrate. The generation of radical species
in the two one-electron reduction steps can result in a
complex profile of reaction products, including dimers,
4. Mechanism of action
4.1. Functional roles
Most reactions catalysed by HRP C and other horseradish peroxidase isoenzymes can be expressed by the
following equation, in which AH2 and AH represent a
reducing substrate and its radical product, respectively.
Typical reducing substrates include aromatic phenols,
phenolic acids, indoles, amines and sulfonates.
HRP C
H2 O2 þ 2AH2 ! 2H2 O þ 2AH
ð1Þ
The conversion of hydrogen peroxide to water is not the
primary function of class III plant peroxidases such as
HRP C. Other enzymes, including ascorbate peroxidase
(class I), are used by plants to regulate levels of intracellular hydrogen peroxide (Mittler, 2002; Shigeoka et al.,
2002). Little is known about the ascorbate peroxidases of
horseradish, but in a recent study the accumulation of
H2O2 in Arabidopsis thaliana plants deficient in cytosolic
Fig. 4. The catalytic cycle of horseradish peroxidase (HRP C) with
ferulate as reducing substrate. The rate constants k1, k2 and k3 represent the rate of compound I formation, rate of compound I reduction
and rate of compound II reduction, respectively.
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N.C. Veitch / Phytochemistry 65 (2004) 249–259
trimers and higher oligomers that may themselves act as
reducing substrates in subsequent turnovers.
The first step in the catalytic cycle is the reaction
between H2O2 and the Fe(III) resting state of the
enzyme to generate compound I, a high oxidation state
intermediate comprising an Fe(IV) oxoferryl centre and
a porphyrin-based cation radical. A transient intermediate (compound 0) formed prior to compound I has
been detected in reactions between HRP C and H2O2 at
low temperatures and described as an Fe(III)-hydroperoxy complex. Molecular dynamics simulations of
these peroxide-bound complexes have been carried out
(Filizola and Loew, 2000). In formal terms, compound I
is two oxidising equivalents above the resting state. The
first one-electron reduction step requires the participation of a reducing substrate and leads to the generation
of compound II, an Fe(IV) oxoferryl species that is one
oxidising equivalent above the resting state. Both compound I and compound II are powerful oxidants, with
redox potentials estimated to be close to +1 V. The
second one-electron reduction step returns compound II
to the resting state of the enzyme. Reaction of excess
hydrogen peroxide with resting state enzyme gives
compound III, which can also be prepared by several
other routes (Dunford, 1999). This intermediate is best
described as a resonance hybrid of iron(III)-superoxide
and iron(II)-dioxygen complexes. A high-resolution
crystal structure of 95% pure compound III published
recently shows dioxygen bound to heme iron in a bent
conformation (Berglund et al., 2002). Models for the
irreversible inactivation of HRP C by peroxides have
been developed in studies with m-chloroperoxybenzoic
acid (Rodriguez-Lopez et al., 1997).
Detailed mechanisms showing the role of the distal
heme pocket residues Arg38 and His42 (conserved in all
members of the plant peroxidase superfamily) in the
formation of compound I have been described in the
literature but are beyond the scope of this review. High
resolution crystal structures of the oxidised intermediates of HRP C confirm the importance of Arg38
and His42 for peroxide catalysis (Berglund et al., 2002).
For example, the ferryl oxygen of compound I is
hydrogen bonded to Arg38 NEH and a water molecule
that is also hydrogen bonded to both Arg38 and His42.
A mechanism for the reduction of compounds I and II
by ferulate has been proposed based on new information from the crystal structure of a ternary complex
formed between ferulic acid and cyanide-ligated HRP C
(Henriksen et al., 1999).
4.3. Aromatic substrate binding sites
Substrate oxidation by HRP C occurs at the ‘exposed’
heme edge, a region comprising the heme methyl C18
and heme meso C20 protons (Table 1). This interaction
site was identified in enzyme inactivation experiments
with alkyl- and phenylhydrazines, sodium azide and
other reactive agents (Ortiz de Montellano, 1992). The
free radical species generated when these are incubated
with HRP C and H2O2 give adducts such as C18hydroxymethyl and C20-meso-phenyl heme derivatives.
What is particularly interesting is the contrast between
the behaviour of HRP C and other heme proteins such
as the globins and cytochromes P450, where modification to the heme iron atom and pyrrole nitrogens
occurs. Substrate access to the oxoferryl centre of HRP
C appears to be hindered by the local protein environment, an example of ‘closed’ heme architecture. This is
one reason why peroxidases are much less effective as
catalysts of oxygen-transfer reactions than cytochromes
P450. Attempts to improve this aspect of the chemistry
of HRP C have been made by site-directed mutagenesis.
In these experiments, substrate access to the oxoferryl
centre of compound I was increased by substituting the
distal residues Phe41 and His42 with smaller amino acid
residues (Newmyer and Ortiz de Montellano, 1995;
Ozaki and Ortiz de Montellano, 1995).
Stable, reversible 1:1 complexes with a variety of aromatic molecules are formed both by resting state HRP
C and some of its ligand-bound derivatives (e.g.
cyanide-ligated HRP C) in the absence of H2O2. Spectroscopic and crystallographic studies have revealed a
detailed picture of the site where these aromatic substrates are located (Veitch and Smith, 2001). For example, when ferulic acid binds to cyanide-ligated HRP C
its aromatic ring is located in a distal site close to the
exposed heme edge and its phenolic acid side-chain is
oriented towards the entrance of the binding site (Henriksen et al., 1999). There are hydrogen-bonded interactions between Arg38 NZH2 and the phenolic and
methoxyl oxygen atoms of ferulic acid and between the
phenolic oxygen and an active site water molecule. The
latter is also hydrogen bonded to the backbone carbonyl
of Pro139 (an invariant residue in the plant peroxidase
superfamily) and the nitrogen atom of the cyanide
ligand. There are hydrophobic interactions with amino
acid side-chains (Phe68, Gly69, Pro139, Ala140, Phe142
and Phe179) as well as heme methyl C18H3 and the
heme meso proton C20H (Fig. 5). The structure and
dynamics of benzhydroxamic acid complexes of HRP C
have also been investigated in detail with site-directed
mutants used to explore the contribution of binding site
residues to substrate affinity (Veitch and Smith, 2001).
5. The isoenzyme question; horseradish and
Arabidopsis peroxidases
5.1. The horseradish peroxidase isoenzyme family
Fifteen peroxidase isoenzymes have been isolated from
horseradish root using classical protein purification
N.C. Veitch / Phytochemistry 65 (2004) 249–259
255
neutral isoenzyme (HRP N) that has not yet been isolated from the plant (Bartonek-Roxå and Holm, 1999).
5.2. Arabidopsis thaliana peroxidases
Fig. 5. The ferulic acid binding site of horseradish peroxidase (HRP
C). This representation is taken from the X-ray crystal structure of the
ternary complex of ferulic acid (FA) and cyanide-ligated HRP C
(Brookhaven accession code 7ATJ). The heme group is shown in red
and ferulic acid, the cyanide ligand (bound to heme iron at the vacant
distal coordination site) and the catalytic residues Arg38, His42 and
His170 are in atom colours. Several phenylalanine side-chains that
contribute to the binding site are shown in blue. Note that the ferulic
acid side-chain is located towards the entrance of the binding site.
methods and are generally referred to by codes based on
their isoelectric point value as HRP A1–A3 (‘acidic’),
B1–B3 and C1–C2 (‘neutral’ or ‘neutral-basic’) and D
and E1–E6 (‘basic’). An even greater number of peroxidase ‘isoforms’ can be detected by isoelectric focusing
but their identity has proved controversial as some may
be artifacts produced by deamidation, cross-linking or
other processes. The fact that amino acid sequences
determined for HRP A2 and E5 show only 54 and 70%
identity, respectively, to HRP C, is one piece of evidence
for the existence of distinct peroxidase isoenzymes in
horseradish root (Welinder, 1992b).
Four genomic DNA clones encoding neutral and
neutral-basic HRP isoenzymes have been isolated and
characterised from cultured cells of Armoracia rusticana
(Fujiyama et al., 1988, 1990). Two of these peroxidase
genes (prxC1a and prxC1b) are expressed both in stems
and roots and two mainly in roots (prxC2 and prxC3).
All consist of four exons and three introns and have
identical splice site positions, a feature common to other
plant peroxidase genes. Three highly homologous
cDNAs (prxC1a, prxC1b and prxC1c) have also been
isolated using an oligonucleotide probe corresponding
to the primary sequence region from His40 to Ala51 of
HRP C (Fujiyama et al., 1988). The amino acid
sequence deduced from prxC1a is identical to that of
HRP C but also contains a hydrophobic leader
sequence as well as a C-terminal extension which may
be responsible for vacuolar targeting. Expression of the
prxC2 gene is induced by wounding and its transcriptional regulation has been investigated in detail (Kaothien et al., 2000). One further cDNA has been cloned
and sequenced from horseradish corresponding to a
The sequencing of the genome of the ‘model’ plant,
Arabidopsis thaliana, and the availability of expressed
sequence tag (EST) databases of cDNA clones has provided the opportunity to investigate an entire family of
class III peroxidases from one plant for the first time,
with striking results. According to recent studies there
are 73 full-length genes for class III plant peroxidases in
the Arabidopsis genome (Tognolli et al., 2002; Welinder
et al., 2002). Of these, 71 are predicted to encode for
stable enzymes folded similarly to HRP C (Welinder et
al., 2002). Amino acid sequence identities among the
predicted peroxidases range from 28 to 94%. Alignment
of these sequences has been used as a basis for comparisons with other plant peroxidases and the identification
of potentially similar enzymes. For example, expression
of an acidic peroxidase isoenzyme from A. thaliana
(AtPA2) with 95% amino acid sequence identity to
HRP A2 (a horseradish peroxidase isoenzyme of
unknown function) was found to coincide with lignification (Østergaard et al., 2000). The three-dimensional structure solved for recombinant AtPA2 by Xray crystallography reveals a substrate binding site that
can accommodate monolignols such as p-coumaryl and
coniferyl alcohols (Nielsen et al., 2001). This structure is
an excellent model for HRP A2, which has not yielded
crystals suitable for crystallographic analysis. Both
enzymes will catalyse the oxidation of monolignols and
phenolic acids although small differences in their reactivity can be detected. These may have their origin in the
levels of glycosylation of the two enzymes; recombinant
AtPA2 (as expressed in E. coli) is non-glycosylated,
whereas plant HRP A2 is highly glycosylated with one
bulky glycan attached close to residues at the entrance
of the substrate binding site.
The presence of similar peroxidase isoenzymes in taxa
from two genera of the same plant family may not seem
surprising, but analysis of Arabidopsis peroxidases indicates that enzymes from species in quite unrelated plant
families may also share high amino acid sequence identity, for example, Arabidopsis peroxidase AtP1 (Cruciferae) and a peroxidase from Gossypium hirsutum
(Malvaceae). Duroux and Welinder (2003) have used
the peroxidase gene family of A. thaliana as a basis for a
comprehensive survey of evolutionary relationships
among class III plant peroxidases from angiosperms,
gymnosperms, ferns, mosses and liverworts. They
suggest that the class III plant peroxidase gene family
appeared when plants colonised the land and that adaptive
advantages were conferred through peroxidase functionality (e.g. in cell-wall metabolism and defence). Phylogenetic analysis confirms that the class III peroxidases of A.
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thaliana represent a highly diverse gene family. Peroxidases from eudicots cluster with the different groups
present in Arabidopsis, indicating that peroxidase diversity was extant before the radiation of eudicots. In contrast, peroxidases from monocots cluster in specific
groups that have not been found in eudicots. It appears
therefore that some groups of peroxidases evolved
independently in monocots and eudicots or that some
were lost. What is remarkable is the inference that
each plant species contains a relatively large number
of distinctive peroxidase enzymes. The challenge for
the future lies in the determination of their specific
functions.
6. Protein engineering and directed evolution of
HRP C
Several expression systems for the production of
recombinant HRP C have been developed since the
early 1990s and are compared in a recent review (Veitch
and Smith, 2001). Active recombinant HRP C in yields
of 5–10 mg/l can be produced in a baculovirus system
although the enzyme is highly glycosylated. Yields are
lower (typically 2–4 mg/l) when Escherichia coli is used
as the expression system because the enzyme must be
recovered from inclusion bodies. Essential steps in this
process are the solubilization of the inclusion bodies,
controlled reoxidation of the reduced, denatured
enzyme and in vitro refolding in the presence of heme
and calcium (Smith et al., 1990). One advantage of E.
coli expression is that active recombinant enzyme is
obtained in non-glycosylated form, a factor crucial to
the success of subsequent crystallisation studies and the
solution of the three-dimensional structure of the
enzyme. Site-directed mutants of the enzyme have been
generated in both baculovirus and E. coli expression
systems. Areas of interest addressed in these studies
include the role of distal heme pocket residues in peroxide binding and catalysis, the identity of structural
determinants of substrate oxidation, characterisation
of small molecule and substrate binding sites, the
significance of calcium binding sites and the rational
design of artificial binding sites. A comprehensive
survey of work published on site-directed mutants of HRP
C up to the end of 1999 is available (Veitch and Smith,
2001) and some results are summarised in Table 1.
Directed evolution techniques have been applied to
HRP C more recently in an attempt to improve qualities
of the enzyme that are important for biotechnological
and diagnostic applications, such as thermal stability
and resistance to inactivation by peroxides. In these
studies the host chosen for expression of HRP C was
Saccharomyces cerevisiae rather than E. coli, as active
enzyme can be produced without the need for in vitro
refolding. However, expression levels in S. cerevisiae are
low (approximately 50 mg/l) and the secreted enzyme is
hyperglycosylated. A 40-fold increase in the HRP C
activity of S. cerevisiae culture supernatant was
achieved in three rounds of directed evolution using
random point mutagenesis and screening (Morawski et
al., 2000). The majority of the mutations were to amino
acid residues in either loop or surface regions of the
enzyme. Although activity was improved, thermal
stability showed a small decrease with respect to wildtype enzyme. However, the combination of an HRP C
mutant evolved for higher activity with a single site
mutation at Asn175 (N175S) gave significant improvements in thermal stability (Morawski et al., 2001).
Expression of this mutant in Pichia pastoris (giving
sufficient amounts of the enzyme for purification and
characterisation) yielded recombinant enzyme showing
a 3-fold improvement in thermal stability at 60 C and
pH 7.0 and increased resistance to inactivation by
hydrogen peroxide and other additives. This recombinant enzyme (‘HRP13A7-N175S’) comprised one
synonymous mutation (A85) and three amino acid substitutions (N175S, N212D and Q223L). In contrast,
directed evolution of a fungal peroxidase from Coprinus
cinereus yielded site-directed mutants with up to 190fold increases in thermal stability and 100 times greater
oxidative stability than the wild-type enzyme (Cherry et
al., 1999). These dramatic improvements must be considered in the light of the greater thermal and oxidative
stability already possessed by wild-type HRP C as
compared to Coprinus peroxidase and the much larger
number of mutants screened for the latter.
7. Horseradish peroxidase and indole-3-acetic acid
7.1. Mechanisms of oxidation
One of the most interesting reactions of HRP C
occurs with the plant hormone, indole-3-acetic acid
(IAA). In contrast to most peroxidase–catalysed reactions, this takes place without added hydrogen peroxide, hence the use of the term ‘indole acetic acid oxidase’
to describe this activity of HRP C in the older literature.
More recent studies of the reaction at neutral pH indicate that it is not an oxidase mechanism that operates,
but rather a peroxidase mechanism coupled to a very
efficient branched-chain process in which organic
peroxide is formed (Dunford, 1999). The reaction is
initiated when a trace of the indole-3-acetate cation
radical is produced. Major products of indole-3-acetic
acid oxidation include indole-3-methanol, indole-3aldehyde and 3-methylene-2-oxindole, the latter most
probably as a result of the non-enzymatic conversion
of indole-3-methylhydroperoxide. Conflicting theories
have been proposed to explain the mechanism of reaction at lower pH (reviewed by Dunford, 1999), in which
N.C. Veitch / Phytochemistry 65 (2004) 249–259
the formation of the ferrous enzyme, compound III and
hydroperoxyl radicals must also be accounted for. The
physiological significance of IAA metabolism by HRP
C and other plant peroxidases is still an area of active
debate. For example, studies of the expression of an
anionic peroxidase in transgenic tobacco plants indicate
that while overproduction of the enzyme favours defensive strategies (such as resistance to disease, physical
damage and insect attack) it has a negative impact on
growth because of increased IAA degradation activity
(Lagrimini, 1996). Thus peroxidase expression in plant
tissues at different stages of development must reflect a
balance between the priorities of defence and growth.
7.2. Targeted cancer therapy
Use of indole-3-acetic acid and HRP C in combination may offer new potential for targeted cancer
therapy, according to recent work by Wardman and
colleagues (Folkes and Wardman, 2001; Greco et al.,
2001; Wardman, 2002). These authors observed that
IAA is cytotoxic towards mammalian cells, including
human tumour cells, in the presence of HRP C. The
primary mechanism of toxicity is thought to involve 3methylene-2-oxindole, a known product of the reaction
between HRP C and IAA that shows high reactivity
towards cellular nucleophiles such as glutathione and
the thiol groups of proteins or histone (Fig. 6). However, the fact that 2-methylindole-3-acetic acid shows
greater cytotoxicity in combination with HRP C than
IAA suggests that there may be other mechanisms of
toxicity which do not involve oxindole intermediates
(Wardman, 2002). Many other substituted indole-3acetic acid derivatives have been tested for cytotoxicity
in combination with HRP C in an attempt to place
relationships between structure and activity on a predictive level. No simple correlation was found between
levels of cytotoxicity of indole derivatives and their
reactivity towards compound I; for example 5-fluoroindole-3-acetic acid is more cytotoxic towards tumour
cells than IAA but less effective as a reductant of compound I (Folkes et al., 2002). Other factors such as the
pKa of the indolyl radical cation and rates of decarbox-
257
ylation and radical fragmentation may also be significant. One of the most cytotoxic indoles identified
from in vitro screens is 6-chloroindole-3-acetic acid, a
derivative with potential as a prodrug for targeted cancer therapies mediated by HRP C (Rossiter et al., 2002).
The challenge now is to develop strategies to evaluate
and implement this promising system in vivo. Indeed
the combination of HRP C and indole-3-acetic acid
or its derivatives offers several advantages for future
antibody-, gene- or polymer-directed enzyme:prodrug
therapies (Folkes and Wardman, 2001; Wardman,
2002). Among the favourable properties of HRP C are
its good stability at 37 C, high activity at neutral pH,
lack of toxicity and the ease with which it can be
conjugated to antibodies and polymers. Furthermore,
evidence available at present suggests that IAA does not
show any adverse side-effects in humans. The fact that
peroxide is not required as a cosubstrate for the reaction
with HRP C is also a significant advantage.
8. Applications overview
Horseradish peroxidase (predominantly HRP C) is
used as a reagent for organic synthesis and biotransformation as well as in coupled enzyme assays,
chemiluminescent assays, immunoassays and the treatment of waste waters (for reviews see Ryan et al., 1994;
Veitch and Smith, 2001; Krieg and Halbhuber, 2003).
Improvements to desirable qualities of the enzyme such
as its relatively good stability in aqueous and nonaqueous solvent systems are actively sought as an outcome of chemical modification, site-directed mutagenesis and directed evolution studies (Ó’Fágáin, 2003).
Some applications of HRP C in small-scale organic
synthesis include N- and O-dealkylation, oxidative
coupling, selective hydroxylation and oxygen-transfer
reactions. Site-directed mutagenesis at Phe41 and His42
of HRP C has been used to improve the enantioselectivity of arylmethylsulfide oxidations (Newmyer
and Ortiz de Montellano, 1995; Ozaki and Ortiz de
Montellano, 1995). However, Van de Velde et al. (2001)
make the general point that scale-up of peroxidase-
Fig. 6. A mechanism proposed for the formation of 3-methylene-2-oxindole from horseradish peroxidase (HRP C) and indole-3-acetic acid (after
Folkes et al., 2002). R represents a cellular nucleophile (e.g. sulphydryl groups of enzymes or histone).
258
N.C. Veitch / Phytochemistry 65 (2004) 249–259
catalysed enantioselective oxidations to industrial level
will require a substantial reduction in the price of
enzyme per unit product. Solutions to this problem
may include better process management of hydrogen
peroxide to avoid enzyme inactivation and use of
engineered enzymes with improved stability and
catalytic efficiency.
Several examples of the use of HRP C in natural
product synthesis or biotransformation have appeared
in the literature. For example, peroxidase-catalysed
oxidative coupling of methyl-(E)-sinapate with the
syringyl lignin-model compound, 1-(4-hydroxy-3,5dimethoxyphenyl)ethanol yielded a novel spirocyclohexadienone together with a dimerization side-product
(Setälä et al., 1999). Coupling of catharanthine and
vindoline to yield a-30 ,40 -anhydrovinblastine is a reaction catalysed by HRP C of potential interest as a
semisynthetic step in the production of the anti-cancer
drugs vinblastine and vincristine from Catharanthus
roseus (Sottomayor et al., 1997). An enzyme with a30 ,40 -anhydrovinblastine synthase activity that shows
many features characteristic of a plant peroxidase has
now been purified from C. roseus leaves (Sottomayor et
al., 1998).
9. Bibliography of horseradish peroxidase
Few plant enzymes are represented so widely in the
scientific and patent literature as horseradish peroxidase. This has limited the number of primary sources
that can be cited in the present report and a bias
towards more recent papers has been adopted. Review
articles with more extensive lists of references are quoted where appropriate. Two important monographs on
peroxidase enzymes have been published by Dunford
(1999) and Saunders, Holmes-Siedle and Stark (1964).
The latter contains approximately 1500 references
covering the period from 1810 to 1960. Two volumes
of reviews and literature compilations on peroxidase
covering the decades 1970–1980 and 1980–1990 were
published by the University of Geneva and contain
more than 5000 references (Gaspar et al., 1982, 1992).
Other general reviews on peroxidase are listed in
Veitch and Smith (2001). Two review articles that deal
specifically with horseradish peroxidase are available
in the literature (Dunford, 1991; Veitch and Smith,
2001).
Acknowledgements
The author thanks Andrew Smith, Karen Gjesing
Welinder and Claude Penel for their generous assistance
during the preparation of this review.
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